This invention relates to diagnostic methods for the detection of Dirofilaria immitis antigens in blood or bodily fluid samples of infected animals and, more particularly, to improved means for achieving rapid separation of Dirofilaria immitis immune complexes in such samples preparatory to carrying out an assay for Dirofilaria immitis infection in animals, notably dogs.
Heartworm disease (dirofilariasis) is caused by Dirofilaria immitis, a filarial nematode parasite primarily of the dog, and is distributed throughout the world. While the parasite can cause heart failure, lung disease, disability and death, infected animals often have no outward evidence of disease. Signs of heartworm disease in dogs, when present, are nonspecific. The diagnosis of D. immitis infection is most commonly made by demonstrating microfilariae, larval forms of the parasite, in peripheral blood smears. However, this timehonored test is insensitive, and it is now well recognized that a significant proporti-on of infected dogs lack microfilaremia.
Recently, Weil (copending U.S. application Ser. No. 557,117, filed Dec. 1, 1983) and Weil et al., The Journal of Immunology, Vol. 134, No. 2, p. 1185-1191 February 1985) has identified circulating parasite antigens of Dirofilaria immitis present in the serum of D. immitis infected dogs and characterized the antigens to the extent necessary to distinguish these antigens from other antigens, and thereby render it possible to detect these specific antigens in the blood or bodily fluids of D. immitis infected animals. Weil has also produced and characterized monoclonal antibodies specific for such circulating D. immitis antigens and has developed a sensitive assay to detect parasite antigenemia in D. immitis-infected dogs. The assay involves providing a sample of blood or bodily fluid from an animal infected with or suspected of being infected with Dirofilaria immitis and assaying for the presence of the circulating parasite antigens of Dirofilaria immitis by means, for example, of a double antibody assay such as the sandwich ELISA assay in which a polyclonal antibody and a monoclonal antibody are used as the first and second antibodies.
Briefly, in one embodiment of the Weil assay, a rabbit polyclonal antibody directed towards the circulating parasite antigens of Dirofilaria immitis is attached to a solid support. The sample to be assayed and a horseradish peroxidase conjugated monoclonal antibody to the antigens is added to the solid support and allowed to react. If antigens are present in the sample, a polyclonal antibody-antigen-conjugated monoclonal antibody sandwich is formed which following addition of a horseradish peroxidase substrate will develop color. This color is subjectively or quantitatively compared to standards by the user and a determination of the presence or absence of the antigen is made.
One major problem in detecting circulating parasite antigens in accordance with Weil's assay is that the host's immunological response to the infection produces specific antibodies to the circulating antigens, thus forming immune complexes. These immune complexes are not detectable in some cases and must be separated in order to free the antigen of interest. In the above-noted Weil et al. publication, the method disclosed for freeing the antigens from the immune complexes involves the addition to the sample of 0.1 M disodium EDTA, pH 7.5, and heating to 100.degree. C. for 5 minutes followed by centrifugation for 5 minutes at 16,000.times.G. Similarly, in Weil et al. Am. J. Trop. Med. Hyg. 33(3), 1984, p. 425-430, separation of the immune complexes involved the addition of 8% polyethylene glycol (PEG) in phosphate buffered saline (PBS) to a serum sample, incubation for 60 minutes at 4.degree. C. and centrifugation at 16,000.times.G for 5 minutes. Similar procedures have been reported for separating complexes in the case of mycotic organisms (Weiner, Journal of Clinical Microbiology, Vol. 18 July, 1983, p. 136-142; Weiner et al., J. Lab. Clin. Med., Vol. 93, January, 1979, p. 111-119; and Weiner, Annals of Internal Medicine, 1980, 92:793-796). While these methods do effect separation of the immune complexes, the employment of higher temperatures such as 100.degree. C. and/or centrifugation causes or tends to cause coagulation of the protein and therefore renders such methods inconvenient or impractical for routine use in veterinary clinics and the like.
There has been a need, therefore, for a rapid, practical, effective and convenient method for effecting separation of Dirofilaria immitis immune complexes in canine serum or other samples preparatory to assaying such samples for the presence of circulating antigens of Dirofilaria immitis.